Analysis of a histological tissue sample is commonly used for diagnosis purposes, e.g. analysis of a breast tissue sample for diagnosing breast cancer, or for research purposes, e.g. to study inflammatory cell types in inflammatory conditions such as asthma, atherosclerosis, or inflammatory bowel diseases.
Immunohistochemistry (IHC), whereby a marker (i.e. an antigen) is detected by a antigen-specific antibody, is commonly used to identify cells in histological sections. Ideally, identification of a cell type can be obtained with detection of one cell-specific cell antigen. However, for several cell types combinations of several antigens must be analysed to for proper identification.
Any diseased tissue is typically associated with an altered cellular composition. For example, in inflamed airways in asthma there is an altered composition of the structural cells that build up the airways, such as epithelial cells, gland cells, blood vessel cells, nerves etc. In addition, several types of immune cells (i.e. leukocytes) infiltrate the inflamed airways.
In many diseases the pathological (i.e. destructive alterations) in the tissue is not caused by one cell type but rather a complex interaction between several cell types. Hence, when exploring a diseased tissue sample it is often desirable to study several cell populations and tissue structures. Information about the cellular content can be obtained by staining one cell type at the time in serially cut sections. Although this approach provides a good estimation of the content of several cell types in a tissue sample, it does not provide detailed information about the spatial relationship (i.e. physical relationship) between the analysed cell types.
In order to better explore how the composition of cells may define certain disease conditions, or study how cells interact and relate to each other inside a diseased tissue, it is desirable to develop means to visualize multiple cell types within the same three dimensional space, for example within one single tissue section.
With currently available IHC techniques it is possible to stain up to 4 cell types in one section using multiple-chromogen or multiple immunofluorescence techniques. In common practice, however, often only 2 cell types can be simultaneously detected due to lack of appropriate combinations of primary detection antibodies.
In order to increase the number of markers in one tissue section, new methodological approaches have been developed, such as the SIMPLE technique disclosed in WO 2010/115089 and the MELC technique (Schubert et al., Nature Biotechnology v. 24, pp. 1270-1278). Although powerful, these new types of techniques have primarily been developed for co-localization studies and either involve tissue-destructive procedures, procedures involving destruction of detection groups, or dependence of detection molecule-labeled primary antibodies, features that limit the number of cell markers that can be stained.
Since the above mentioned techniques were primarily developed for co-localization studies they do not deal with the fact that many identification markers may occasionally also be present on non-intended cell types.
Hence, there is a need for new technical approaches by which a large number of cell types can simultaneously be properly identified within the same physical space, such as one tissue section. Ideally, any such technique should be capable of analysing an entire large section of samples and providing detailed information about all marked individual cells such as their spatial coordinates in the tissue, their size and shape parameters etc.